Metastasis and Tumor Dormancy
Early diagnosis of neoplastic disorders such as breast cancer, melanoma and renal cancer, can increase the chances of disease-free survival of patients. However, these disorders can recur as metastatic disease many years after primary tumor resection and adjuvant therapy. This metastatic disease appears to arise from tumor cells that disseminated early in the course of the disease but did not develop into clinically apparent lesions, and is the major cause of mortality of breast cancer patients.
These long-term surviving, disseminated tumor cells maintain a state of dormancy and are resistant to conventional therapies that target actively-dividing cells, but may be triggered to proliferate through largely unknown mechanisms. Therefore, understanding the mechanisms that regulate tumor dormancy or the switch to a proliferative state is critical to discovering novel targets and interventions to prevent disease recurrence.
Chemotherapy is the main treatment for disseminated, malignant cancers. However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. This failure is in part due to the intrinsic or acquired drug resistance of many tumor cells. Another drawback to the use of chemotherapeutic agents is their severe side effects. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth.
In addition, currently available cancer therapy commonly targets actively proliferating tumor cells, and fails to eradicate quiescent, non-proliferating tumor cells (dormant tumor cells) which are the source for the recurrent disease and metastasis.
Two states of tumor dormancy have been described in the literature based on experimental and clinical evidence. Dormant tumor cells may exist in a quiescent state for many years as solitary quiescent tumor cells in the bone narrow, lymph nodes and blood circulation of breast cancer patients. Their quiescence was demonstrated by their negative staining for the proliferation marker Ki67 and their negative staining for apoptosis. These cells are resistant to conventional therapies that target actively dividing cells, leading to possible disease recurrence following adjuvant therapy.
Alternatively, tumor dormancy may exist as micrometastases where cellular proliferation is balanced by apoptosis. Consequently, in this balanced state, there is no net increase in tumor mass over time. These micrometastases remain dormant because of lack of recruitment of the vasculature needed to nourish the tumor, known as the angiogenic switch and/or involvement of the adaptive immune system such as cytotoxic CD8+ T cells.
Macrophage Types
Macrophages are highly plastic monocyte-derived cells that acquire different molecular and functional phenotypes following exposure to different bioactive molecules and environments. The early studies on the interactions of macrophages and lymphocytes in battling bacterial infections revealed the T helper type 1 (Th1) secreted cytokine IFNγ to be involved in the classical activation of macrophages. However, seminal studies by the groups of Gordon and Mantovani have extensively characterized additional macrophage subtypes activated in alternative manners (reviewed in Mantovani et al., 2004, Martinez et al., 2009).
Since the major polarizing cytokines initially found to be involved in classical and alternative activation were derived from Th1 (IFNγ) and Th2 (IL-4 and IL-13) lymphocytes these activated macrophages were named M1 and M2, respectively. Later studies revealed that, in addition to IL-4, alternative activation can also be induced by immune complexes and glucocorticoids, and accordingly the subdivision of alternatively-activated macrophages to M2a-c was instilled. M1 macrophages are important inducers and effectors in the Th1 response. They are instrumental in immune responses against intracellular microbes and tumors. M2 macrophages are more heterogeneous, but generally play a role in Th2 responses, such as killing and encapsulation of extracellular parasites, stopping inflammation and promoting tissue repair and remodeling. M2 macrophages also play a role in immune regulation and promote tumor progression (Mantovani et al., 2005, Martinez et al., 2009). M1 and M2 macrophages are not only distinct in function, but also express different receptors and enzymes required for their activities. M1 macrophages express high levels of inflammatory cytokines (IL-12, IL-23, TNFα, IL-1β, and IL-6) and chemokines (CXCL9, 10, and 11, CCL2, 3, 4, and 5, and CXCL2), as well as enzymes involved in the generation of reactive oxygen species (ROS) and nitric oxide (NO). M2 macrophages express lower levels of inflammatory mediators, but high levels of IL-10, scavenger, mannose, and galactose receptors.
The prototypic Th2 cytokines IL-4, IL-13 and IL-10, as well as immune responses to parasites were found to promote many of the outcomes of efferocytosis (the process by which dying or dead cells are removed by phagocytic cells) in macrophages. These cytokines are well appreciated antagonists of the M1 response and macrophage pro-inflammatory properties while IL-4 and IL-13 can also promote fibrosis through TGFP production. IL-4 and IL-13 also activate PPAR-λ and PPAR-δ to promote monocyte/macrophage alternative activation. Liver X receptor (LXR) was recently found to synergize with IL-4 in the induction of arginase 1 expression and promotion of an M2 phenotype in regressive atherosclerotic lesions. Thus, efferocytosis induces phenotypic and molecular switches and activates signaling pathways in macrophages that resemble M2 polarization. In addition, M2 polarization promotes efferocytosis through induction of different molecular modules, whereas M1 macrophages exert reduced uptake of apoptotic cells. Along these lines, recent studies also found that efferocytosis is a self-promoting process, and that M2 pathways play key roles in mediating this feature of macrophage function.
Role of Macrophages in Inflammation
When inflammation occurs, polymorphonuclear neutrophils (PMNs) are among the first leukocyte responders to accumulate in the inflamed site. These cells are crucial as the first line of defense of the innate immune system because of their phagocytotic and microbicidal functions. The initial accumulation of neutrophils is followed by a second wave of cellular infiltration, of mononuclear phagocytes (monocytes). Differentiation of monocytes into macrophages promotes the removal of apoptotic neutrophils and debris by nonphlogistic phagocytosis. Notably, the apoptotic neutrophil uptake blocks the release of pro-inflammatory mediators (including chemokines, cytokines and lipid mediators) from M1 macrophages, in a phenomenon termed reprogramming/“immune silencing”, and promotes production and release of anti-inflammatory and reparative cytokines (M2 macrophages). These M2 macrophages promote myofibroblast proliferation, support matrix deposition, and express inhibitors of metalloproteinase that impairs the remodeling of deposited ECM (Ariel et al., 2012). Therefore, the milieu that governs macrophage differentiation along the M1-M2 axis may dictate the magnitude of myofibroblast activation and deposition of ECM.
Extra Cellular Matrix (ECM) as a Regulator of Tumor Dormancy
In recent studies, the inventors have clarified potential mechanisms by which the ECM, which is part of the microenvironment milieu surrounding the dormant tumor cells, may regulate tumor dormancy and its outbreak to growing metastases (Barkan et al., 2008, 2010 and 2010a). Specifically, it was shown that ECM composition may play a critical role in determining whether solitary dormant tumor cells remain quiescent or begin to actively proliferate, using the well-characterized D2.0R/D2A1 mammary cell line model system to study dormant vs. metastatic proliferative growth. These cell lines were derived from tumors arising from implants of the same D2 hyperplastic alveolar nodule line, but display distinct metastatic properties. Whereas disseminated D2A1 cells in mice transition from dormancy to metastatic growth after few weeks, disseminated D2.0R cells remain dormant for months with occasional formation of metastatic lesions. Using a modified 3D culture system constituted from growth factor reduced basement membrane (BME; a specialized form of ECM), the inventors demonstrated for the first time that the quiescent or proliferative behavior of these cells could be recapitulated in vitro (Barkan et al., 2010a; Barkan et al., 2011). Thus D2.0R cells persist in a quiescent state for at least 14 days in the 3D BME system and only few cells occasionally emerge from dormancy after this period. D2A1 cells, on the other hand, remain quiescent for only 4-6 days in the 3D BME system and then begin to proliferate extensively. Importantly, the quiescent state is characterized by growth arrest associated with expression of the negative cell-cycle regulators p27 and p16, and is not due to a balance between cell proliferation and apoptosis. Notably, this 3D model system was validated with additional cell lines demonstrating dormant and metastatic behavior in vivo. Hence, the model system provides the first in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.
Characterization of Fibrotic Tissue
Fibrosis is one of the pathological features of metastatic outbreak and is a major pathological feature of many other diseases. Fibrosis can lead to permanent scaring, organ malfunction and, ultimately, death, as seen in end-stage liver disease, kidney disease, idiopathic pulmonary fibrosis and heart failure. There are some drugs aimed to treat fibrotic disease, such as corticosteroids (to decrease Col-I synthesis) and angiotensin blocking agents (for inhibition of collagen synthesis and TGFβ1). Unfortunately, none of them has the ability to cure fibrosis.
Excessive deposition of matrix proteins such as Col-I and fibronectin and α-smooth muscle actin (α-SMA) expression in differentiated fibroblasts are the hallmarks of fibrotic processes. Fibrosis is a complex and tightly orchestrated phenomenon; it involves multiple signals mediated by various stromal cells, such as myofibroblasts (which differentiate from fibroblasts), endothelial cells and macrophages.
Fibroblasts are considered to be the primary source of the reparative matrix in all tissues. In response to tissue injury, they proliferate, migrate to the site of injury, and differentiate into their activated form, myofibroblasts. Following differentiation, myofibroblasts acquire an increased contractile ability and are characterized by the expression of α-SMA positive phenotype. In wound healing, these myofibroblasts mediate wound contraction and the formation of a collagen-rich extracellular matrix. Increased activation and proliferation of resident fibroblasts at the wound edge is therefore an important early step that is central to the wound healing process.
The cytokine transforming growth factor-β1 (TGFβ1) is a mediator of tissue repair and wound healing and is also implicated in progressive tissue fibrosis. In addition to its effect on extracellular matrix turnover, TGFβ1 is known to have direct effects on cell phenotype, including the induction of a contractile phenotype and the up-regulation of α-SMA both in vitro and in vivo.
The myofibroblast, by virtue of its ability to express high levels of cytokines, extracellular matrix and α-SMA, is expected to have key roles in inflammation, connective tissue deposition, and lung tissue mechanics, respectively, but that can severely impair organ function when contraction and ECM protein secretion become excessive, such as in fibrosis.
Fibrosis occurs upon deregulated and exaggerated tissue repair that fails to subside and resolve. During active resolution of inflammation, specific signals down-regulate macrophage activation and promote the clearance of activated macrophages, either by apoptosis or by migration through the lymphatic drainage. Disruption of any of these processes can lead to chronic persistent inflammation and fibrosis.
Fibrotic-Like Microenvironment Promotes Dormant Tumor Cells Proliferation
A recent report by Barkan and colleagues has demonstrated for the first time that remodeling of the ECM can regulate the switch of dormant tumor cells to their metastatic outgrowth. Specifically, it has been shown that metastatic D2A1 cell line transitioned from a quiescent state to proliferation upon production of fibronectin in vitro (Barkan et al., 2008). Furthermore, metastatic lesions arising from D2A1 cells in vivo were associated with significant deposition of fibronectin and type I Collagen (Col-I), whereas a related dormant D2.0R cell line did not express fibronectin or Col-I. However, supplementing the 3D BME system with fibronectin and or Col-I induced the transition of D2.0R cells from quiescence to growth. Furthermore, it has been recently demonstrated by the inventors for the first time that the induction of fibrosis, with deposition of Col-I in the in vivo metastatic microenvironment, induced dormant D2.0R cells to form proliferative metastatic lesions (Barkan et al., 2010a). Hence, D2A1 cells were able to escape tumor dormancy by inducing a stromal fibrotic-like response in vivo, whereas D2.0R cells required an exogenous fibrotic stimulus to initiate their proliferative response.
Importantly, these findings are consistent with several clinical observations demonstrating a correlation between fibrosis and breast cancer recurrence (Hasebe et al., 2002). Therefore, a fibrotic-like microenvironment, which can either be induced externally or be associated with the pathology induced by the residing tumor cells, may provide the permissive microenvironment triggering the transition from dormancy to metastatic growth.
Immunomodulating therapies designed to affect various immune cells which may positively or negatively affect tumor progression are also being considered as potential modulators in the treatment of cancer and other pathologies.
WO 1999/029345 discloses a method for the inhibition of angiogenesis in a cell population in a mammal by inhibiting a host cell angiogenic effect in said mammal, optionally by creating for said cell population an environment substantially free of activated macrophages. The method is said to be useful for the treatment of cancer. Other publications based on inhibiting or eliminating macrophages are disclosed in US 2009/258025 and WO 2010/091206.
EP 0211684 discloses a method of promoting tumoricidal activity of cells of macrophage lineage, comprising the step of subjecting the cells of macrophage lineage to an effective amount of a granulocyte-macrophage colony stimulating factor.
US 2003/0108534 discloses a method of treating cancer, comprising administering to a patient in need an effective amount of macrophages produced by culturing monocytes in vitro, said macrophages having at least one of the following properties: their cytotoxic activity without IFN-γ is increased with respect to standard macrophages; their cytotoxic activity is increased with IFN-γ with respect to standard macrophages; and deactivation of the cytotoxic activity following activation of IFN-γ is defined by residual cytotoxic activity compared to the maximum cytotoxic activity presented by the macrophages due to IFN-γ activation.
Macrophages have also been reported to be involved in anti-cancer effects induced by Mycobacterium bovis bacillus Calmette-Guérin immunotherapy of bladder cancer (Luo and Knudson, 2010).
Thus, a number of distinct macrophages phenotypes may exist in the mammalian body during different stages of inflammation and pathology and during steady state homeostasis. Different macrophage populations have been suggested to exert opposing effects on tumor progression and inflammation. In addition, interactions between the macrophages and their environment, such as the inflammatory site, may also influence the phenotype and hence the function of the macrophages. It is therefore considered difficult to determine appropriate parameters for using macrophages for controlled, predictable therapy, and indeed their clinical use is currently limited.
None of the prior art discloses or fairly suggests that isolated macrophage populations may be used for preventing the recurrence of cancer and metastatic progression in patients having cancer remission. There remains an unmet medical need for providing effective and safe treatments for the treatment of cancer, and especially for treating patients in cancer remission against tumor recurrence. Identification of isolated macrophages and secretory products thereof for inducing cell death in dormant, non-proliferating cells would be highly advantageous.